Radiotherapy Induces Aortic Valve Calcification via Re-activation of Human Endogenous Retroviruses

Background: Cardiovascular disease after adjuvant thoracic radiotherapy has become a leading nonmalignant cause of death in cancer survivors. Human endogenous retroviruses (HERVs) are evolutionary relicts from retroviral infections causing integration of their genome into the human germline millions of years ago. These elements can be re-activated through epigenetic changes, induced by, e.g., radiation. In this study we hypothesized that radiation induces transcription of human endogenous retroviruses (HERVs), thereby activating innate immunity (Toll-like receptors, TLR).

Methods: Valvular interstitial cells (VICs) isolated from human aortic valves were subjected to irradiation with subsequent bulk RNA sequencing. Irradiated cells were analyzed for HERV expression, inflammatory and osteoblastic gene expression, as well as for calcific nodule formation. For mechanistic analyses, experiments were repeated in human fibroblasts in which TLRs were knocked out using CRISPR-Cas9, or in which HERVs were either knocked down or overexpressed. ApoE−/− and ApoE−/−/Tlr3−/− mice were subjected to thoracic irradiation. Aortic valve function was analyzed using transthoracic echocardiography, while leaflet thickness and calcification were evaluated histologically.

Results: Radiotherapy resulted in activation of TLR3, and induction of inflammatory and osteoblastic gene expression in VICs. Irradiation-induced inflammation and osteogenesis were inhibited by genetic deletion of Tlr3. The amount of dsRNA was significantly increased in irradiated cells. Irradiation resulted in re-expression of canonically silenced HERVs. Genetic overexpression of HERVs in VICs induced inflammation and calcification, whereas knockdown of HERVs attenuated both inflammation and calcification after irradiation. Irradiation induced TLR3 expression and aortic valve calcification in ApoE−/− mice. Interestingly, Tlr3-deficient mice were protected from increased leaflet thickness and reduced of aortic valve function after irradiation.

Conclusion: TLR3 induces aortic valve calcification after irradiation via the upregulation of HERVs and therefore represents a novel target to avoid the long-term cardiotoxic effects of radiotherapy.